The effect of protein kinase C (PKC) on carbachol (CCh)-activated nonselective cationic current (ICCh) was investigated in guinea-pig gastric myocytes using a PKC activator, phorbol 12, 13 dibutyrate (PDBu). Pretreatment with 1 micro M PDBu suppressed ICCh by 96.5 +/- 2.9% (n = 14) in a reversible manner in nystatin-perforated mode. In the presence of 1 microM chelerythrine, a PKC inhibitor, inhibition of ICChby PDBu was not seen. In whole-cell mode, the inhibition of ICCh by PDBu was dependent on intracellular MgATP. In the presence of MgATP in the pipette, PDBu decreased ICCh by 98.8 +/- 1.2% (n = 5) as was observed in nystatin-perforated mode. However, PDBu had little effect on ICCh in the absence of MgATP in the pipette; the extent of inhibition was 12.7 +/- 4.3% (n = 8). PDBu also suppressed the generation of cationic current induced by intracellularly perfused GTP[gammaS]. In the PDBu-pretreated group (n = 9) and PDBu-untreated control group (n = 6), GTP[gammaS]-induced currents were 6.7 +/- 2.4 pA and 236 +/- 23 pA, respectively. These results suggest that PKC modulates ICCh at postreceptor sites via protein phosphorylation.